Method for diagnosing lactose intolerance and diagnostic kit for performing the method

ABSTRACT

The method for diagnosing lactose intolerance in patients by the oral administration of defined amounts of lactose, followed by assaying metabolites of lactose, is performed in a way where the lactose administered contains 99%  13 C-labeled lactose, preferably from 5 to 30 mg, and after the administration of the lactose, the, content of  13 C-labeled lactose is determined in a serum sample taken at a defined time after the administration. A diagnostic kit for performing the method consists of a capsule with from 5 to 30 mg of 99%  13 C-labeled lactose, a patient instruction manual, a blood sampling device, sample containers for collecting the blood sample, and optionally a spatula and sample container for a stool specimen.

[0001] The present invention relates to a method for diagnosing lactoseintolerance in patients by the oral administration of defined amounts oflactose, followed by assaying metabolites of lactose, and a diagnostickit for performing the method.

[0002] Lactose intolerance is a universally very frequent phenomenon andresults in a more or less severe digestive disorder from milk and dairyproducts in the afflicted persons. Lactose intolerance is generallybased on the absence of the enzyme lactase for whose formation a gene onchromosome 2 is responsible. This gene determines the time from whichhumans no longer produce lactase. Depending on the ethnic origin, thiscan be the case sooner or later. In earlier times of evolution, humans,like many other mammals, lost the enzyme always when they were no longerfed with breast milk (cf. Pharm. Ztg. No. 9, 145th annual volume, Mar.2, 2000). Today, the H₂ breath test is employed most frequently fordiagnosing lactose intolerance since it is simple and telling. Thus, thepatient drinks a solution of 50 g lactose in water. The hydrogensubsequently exhaled is then measured repeatedly by gas chromatographyover 4 hours. Thus, not only qualitative, but also quantitativeinformation about the lactase deficiency is obtained.

[0003] Lactose intolerance is now considered a disease which can betreated only symptomatically to date. However, in the meantime, therehas been found a possibility of ingesting the enzyme lactase in the formof capsules, tablets or solution and thus compensating the lactasedeficiency.

[0004] In addition to the hydrogen in the breathing air as alreadymentioned, there is also a lactose tolerance test in which 50 g oflactose in water is administered to subjects on empty stomachs, followedby measuring the blood glucose level in such subjects over severalhours. Unless the lactose is completely cleaved enzymatically, theglucose level remains low and thus confirms the lactose intolerance.

[0005] The diagnostic methods known to date have the drawback that arelatively high amount of lactose must be delivered to the patients,which may already lead to considerable complaints in many patients whosuffer from lactose intolerance.

[0006] The assaying of the blood glucose level has the further drawbackthat the blood glucose level may be changed by secondary factors, forexample, increased release of adrenalin due to stress.

[0007] Both prior art methods have the further disadvantage that themeasurements must be performed with several samples over an extendedperiod of time, which is inconvenient and stressing for the patient andadds to the costs.

[0008] In theory, it would be possible to perform the test with¹³C-labeled lactose. However, one gram of ¹³C-labeled lactose costs U.S.$2,000. For the lactose breath test, at least 150 mg of 99% ¹³C-labeledlactose is needed.

[0009] It was the object of the invention to provide a better, lessexpensive and more precise method for diagnosing lactose intolerancewhich does not have the above disadvantages.

[0010] According to the invention, this object is achieved by the oraladministration of defined amounts of lactose, followed by assayingmetabolites of lactose, wherein the lactose administered contains 99%¹³C-labeled lactose, i.e., preferably in amounts of from 5 to 30 mg.After the administration of the ¹³C-lactose, the *content of ¹³C-labeledlactose is determined in a blood sample taken at a defined time afterthe administration. Preferably, the ¹³C-lactose is administered in theform of a gelatin capsule. Thus, it is possible that the ¹³C-lactosereaches the small intestine without being absorbed in the stomach(enteric dosage form).

[0011] In addition, the method can be improved in expressiveness byadditionally determining the content of ¹³C-labeled lactose and/or¹³C-labeled lactic acid in a

[0012] In addition, the method can be improved in expressiveness byadditionally determining the content of ¹³C-labeled lactose and/or¹³C-labeled lactic acid in a stool specimen obtained some time after thetime of the blood sampling. This additional determination is necessaryonly if the examination of the serum sample for glucose does not show asignificant increase of ¹³C and thus the lactose intolerance hasactually been established already.

[0013] Patients with a very pronounced lactose intolerance can be causedto suffer from diarrhoea by a reduced amount of as low as 5 to 10 g oflactose. In this case, such stool specimens still contain almost theentire amount of ¹³C-labeled lactose. In patients with a less pronouncedlactose intolerance, it is possible for the enteric bacteriatometabolize the administered lactose wholly or partially into ¹³C-labeledlactic acid. In addition, in such cases, ¹³C-labeled methane and H₂ mayalso be formed; these are substantially more difficult to examine,however. A decrease of the pH values of the stool specimens from lacticacid is another indication of lactose intolerance. However, the pH valueof the stool may be influenced by many other factors, so that thisparameter is hardly suitable for diagnosing lactose intolerance withcertainty.

[0014] In contrast to the previously employed diagnostic methods, themethod according to the invention requires a significantly smalleramount of lactose, namely from 5 to 30 mg of ¹³C-lactose in one capsule.In patients with a severe lactose intolerance, this smaller amount doesnot lead to any complaints while the diagnostic method is performed.

[0015] However, it is of critical importance that the method accordingto the invention is essentially more precise and, in addition, moretelling because it is not interfered with by secondary influences. Thisis true of the H₂ breath test, and to a much larger extent of thedetermination of glucose in the serum.

[0016] The determination of the ¹³C content in the serum samples and/orstool specimens according to the invention is effected, in particular,by means of modern mass spectrography with previously performed gaschromatography or elemental analysis. In the meantime, these methodshave been further developed and automated to such an extent that it iseconomically possible to send the samples from the patients to a centrallaboratory which has the appropriate equipment and have them examinedthere.

[0017] The central laboratory should analyze only the serum samples inthe first place. If stool specimens are also sent in, these should beexamined only if the corresponding serum sample did not exhibit asignificant rise of the ¹³C-labeled glucose. When the ¹³C-content risesnormally as compared with standard samples of a known ¹³C content, it isalready indicated that lactose is well tolerated and sufficientlydecomposed into glucose by lactase. Only if there is no rise of the ¹³Ccontent, or a significantly lesser one, it can be established byexamining the stool specimen whether the lactose intolerance is verysevere or moderate.

[0018] If the test yields a normal increase of the ¹³C content and yetmilk and dairy products are poorly tolerated, this is due to otherdiseases than the wide-spread lactose intolerance. Thus, the methodaccording to the invention may also be employed for differentialdiagnostics. Such a group of patients cannot be helped by make themingest lactase preparations.

[0019] The method according to the invention is further illustrated bythe following Example.

EXAMPLE

[0020] Before the beginning of the test, 0.3 to 0.5 ml of capillaryblood is withdrawn from the subject's finger or earlobe on an emptystomach. Then, the subject ingests a gelatin capsule with 5 to 30 mg of¹³C-labeled lactose. After 30 to 60 min from the ingestion of thelabeled lactose, from 0.3 to 0.5 ml of blood is again withdrawn asdescribed above. Serum is obtained from the blood samples bycentrifugation. By means of a filter, higher molecular weight components(e.g., lipids and proteins) are removed from the serum samples. Thefiltrates are combusted for an elemental analysis. The CO₂, which isformed thereby from glucose, inter alia, is passed into an isotope massspectrometer by which the ¹³C content is determined. As described above,both blood samples are evaluated. If a difference value Δδ of less than1% is detected between the blood samples taken before and after theingestion of ¹³C-lactose, there is lactose intolerance.

[0021] The test is performed according to the following scheme:

[0022] The patient takes a sample from his next stool and puts it into asample container with a spatula. The next defecation is generallyperformed from 2 to 4 hours after the ingestion of the capsule. From thestool specimens, an aqueous extract is prepared in the laboratory andexamined for ¹³C-lactose and/or ¹³C-lactic acid. The presence of¹³C-lactose or ¹³C-lactic acid confirms the presence of a lactoseintolerance.

1. A method for diagnosing lactose intolerance in patients by the oraladministration of defined amounts of lactose, followed by assayingmetabolites of lactose, characterized in that the lactose administeredcontains 99% ¹³C-labeled lactose, and after the administration of the¹³C-lactose, the content of ¹³C-labeled lactose is determined in a serumsample taken at a defined time after the administration.
 2. The methodaccording to claim 1, characterized in that said ¹³C-labeled lactose isadministered in a capsule.
 3. The method according to claim 1 or 2,characterized in that the content of ¹³C-labeled lactose and/or¹³C-labeled lactic acid is additionally determined in a stool specimenobtained some time after the time of the serum sampling.
 4. The methodaccording to any of claims 1 to 3, characterized in that the amount of99% ¹³C-lactose administered is from 5 to 30 mg, and the serum sample iswithdrawn about one hour after the administration of the lactose.
 5. Themethod according to claim 3 or 4, characterized in that said stoolspecimen is taken 1 to 5 hours after the serum sample.
 6. The methodaccording to any of claims 1 to 5, characterized in that the samples aresent to a central laboratory where they are examined for their ¹³Ccontent by mass spectrography with previously performed gaschromatography.
 7. A diagnostic kit for performing the method accordingto any of claims 1 to 6, consisting of: 1) from 5 to 30 mg of 99%¹³C-labeled lactose in a capsule; 2) a patient instruction manual; 3) ablood sampling device; 4) a sample container for collecting the bloodsample; and optionally 5) a sample container and spatula for a stoolspecimen.